Fluorogenic dendrimer reporters and related methods of use

Abstract

The present invention relates to fluorogenic dendrimer reporters. In particular, the present invention relates to dendrimer nanoparticles conjugated with ‘click-on’ fluorogenic reporters and related methods of use.

Description

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0001]This invention was made with government support under W911NF-07-1-0437 awarded by the U.S. Army Research Office. The government has certain rights in the invention.FIELD OF THE INVENTION[0002]The present invention relates to fluorogenic dendrimer reporters. In particular, the present invention relates to dendrimer nanoparticles conjugated with ‘click-on’ fluorogenic reporters and related methods of use.BACKGROUND OF THE INVENTION[0003]Understanding dynamic cellular processes requires the ability to track biomacromolecules and small molecule metabolites in complex biological environments including cells and animal models. Traditionally, molecules have been tracked using genetically encoded fluorescent labels like GFP or with exogenous antibody reporters (see, e.g., Kulkarni, G.; Wadsworth, W. G. Nat Methods 2012, 9, 451; Prescher, J. A.: Contag, C. H. Curr Opin Chem Biol 2010, 14, 80; Hoffman, R. M. Method Enzymol 2...

AI Technical Summary

Benefits of technology

[0005]The application of small molecule fluorescent reporters to monitor biological systems is limited by their poor water solubility and background fluorescence of these reporters. To address such limitations, the present invention provides dendrimer nanoparticles conjugated with a ‘click-on’ fluorogenic reporter (e.g., 3-azido coumarin, 9-azido anthracene, 6-azido napthalimide, 7-ethynyl coumarin, 2-ethynyl benzothiazole, and 6-ethynyl napthalimide) thereby generating fluorogenic dendrimer reporters. Such fluorogenic dendrimer reporters are not limited to particular uses. In some embodiments, such fluorogenic dendrimer reporters (e.g., dendrimers conjugated with a ‘click-on’ fluorogenic reporter (e.g., 3-azido coumarin, 9-azido anthracene, 6-azido napthalimide, 7-ethynyl coumarin, 2-ethynyl benzothiazole, and 6-ethynyl napthalimide)) are used to track functionalized small molecules configured to interact with the ‘click-on’ fluorogenic reporter to develop a triazole ring, thereby permitting the ‘click on’ fluorogenic reporter to fluoresce. As such, in embodiments wherein the ‘click-on’ fluorogenic reporter is azido based (e.g., 3-azido coumarin, 9-azido anthracene, 6-azido napthalimide), the functionalized small molecule is an alkyne functionalized small molecule (e.g., wherein the azido interacts with the alkyne to develop a triazole ring thereby permitting the ‘click-on’ fluorogenic reporter to fluoresce). In some embodiments, the alkyne within the alkyne functionalized small molecule is an alkyne group. In some embodiments, the alkyne within the alkyne functionalized small molecule is a cycloalkyne group. Similarly, in embodiments wherein the ‘click-on’ fluorogenic reporter is alkyne based (e.g., 7-ethynyl coumarin, 2-ethynyl benzothiazole, and 6-ethynyl napthalimide), the functionalized small molecule is an azido functionalized small molecule (e.g., wherein the alkyne interacts with the azido to develop a triazole ring thereby permitting the ‘click-on’ fluorogenic reporter to fluoresce). Thus, fluorescence from the fluorogenic dendrimer reporters is quenched until it binds with the respective functionalized small molecule (e.g., generating a triazole ring and resulting in detectable fluorescence) thereby permitting the tracking and monitoring of the small molecule and any biological process associated with such small molecule.

[0006]Experiments conducted during the course of developing embodiments for the present invention describe the synthesis and testing of a fluorogenic ‘click’ dendrimer reporter to monitor cellular processes. The reporter system consists of a polyamidoamine (PAMAM) dendrimer conjugated with 3-azido-7-hydroxy coumarin. After the copper (I)-catalyzed azide-alkyne cycloaddition reaction (‘click’ reaction) with alkyne-derivatized target molecules, the natively non-fluorescent construct was shown to have a strong enhancement in fluorescence. This fluorogenic dendrimer reporters were shown to be used to efficiently monitor biological processes and the specificity afforded by the ‘click’ reaction greatly reduced background noise and enhanced assay flexibility. Moreover, such experiments demonstrated that the fluorogenic dendrimer reporters (e.g., dendrimers conjugated with 3-azido coumarin molecules) have (in comparison to non-dendrimer based reporters utilizing coumarin) (1) improved aqueous solubility, (2) the ability to tune signal-to-noise with reduced background fluorescence in biological matrices, and (3) enhanced assay throughput owing to the reductions in purifications steps.

Problems solved by technology

The application of small molecule fluorescent reporters to monitor biological systems is limited by their poor water solubility and background fluorescence of these reporters.

Images